Catalyst: 3-17Catalyst: 3-17

What is gene therapy? How can it help What is gene therapy? How can it help people?people?

Genetic DiseasesGenetic Diseases

Medical conditions caused by an Medical conditions caused by an error in a person’s genetic materialerror in a person’s genetic material

Some Show as Birth DefectsSome Show as Birth Defects Others do not become evident until Others do not become evident until

childhood or adult life. childhood or adult life. Can be mild (color blindness) to life Can be mild (color blindness) to life

threateningthreatening

Gene Therapy• Gene therapy refers to treating genetic disorders

by correcting a defect in a gene or by providing a normal form of a gene.

This Little Light This Little Light of Mine:of Mine:

Transform bacteria with a Jellyfish

gene to make them glow

Aequorea victoria: Source of “glowing gene” for this experiment

Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters

HOW DOES THIS HAPPEN???

DNA TECHNOLOGY!!!

• Recognize specific base sequences in DNA• Cut DNA at those recognition sites

Restriction Enzymes

Recombinant DNA: combination of DNA from 2 or more sources

Put Recombinant into bacteria cells and clone them… then put them

where you want them!!!

SCIENCE IN THE NEWS: Seg 7

• What is manic depression?

• What is its connection to the human genome project?

• How might the discovery of a genetic cause for manic depression help patients like Kay Redfield Jamison, the woman featured in the segment?

• According to Francis Collins, the second scientist interviewed, why are scientists excited about the Human Genome Project?

STUDY GUIDES• In Groups• Work on page 64 Diagram first off• Then work on the rest of that section

DNA Fingerprinting

Determination of an individual’s unique collection of DNA restriction fragments

Collect Tissue Sample

How to do DNA FingerprintingThe Big Picture

>1000 cells

RFLP / Southern blot PCR AnalysisRFLP / Southern blot

>20 cells

RFLP Analysis

RFLP – Restriction Fragment Length Polymorphism; for related DNA molecules, a difference in DNA fragment sizes after restriction enzyme digestion– Difference results from presence of different

DNA sequences– Certain regions of genome are highly variable

AGATCTWild-type allele

Mutant allele

TCTAGA

A single nucleotide change can make a difference

AGAGCT

TCTCGA

Restriction site

Not a restriction site

Example: Sickle-cell allele destroys an MstII site

Need to Analyze only a Small Fraction of Genome

• Human genome is too big to analyze:

• 3 x 109 base pairs 65,536 bp between cuts

= ~46,000 bands

• Most regions of genome are not suitable:

• 99.9% of DNA sequence is same from one person to the next

• Solutions:

• Limit analysis to a few genomic regions

• Focus on regions which are highly variable

How to Focus on Specific Regions of Genome

Need a probe:A short single stranded DNA which is complementary to the region of interest

CAGTATACACAAGTACCGTACCTGGCTCAGTTATACGCCGA

A probe will base pair to the region of interest

GTCATATGTGTTCATGGCATGGACCGAGTCAATATGCGGCT:::::::::::::::::::::::::::::::::::::::::

ATGGCATGGACC::::::::::::

probe

Southern Blotting

Simple Tandem RepeatsSimple Tandem Repeats(STRs)(STRs)

Simple Tandem Repeats(STR’s)

STR – region of DNA containing tandem copies of di-, tri- or tetranucleotide repeat units.

Examples:Dinucleotide repeats: GTGTGTGTGTGT……

Trinucleotide repeats: ACGACGACGACG……

Tetranucleotide repeats:TATCTATCTATC……

More on STRs

• Number of repeats varies greatly between individuals

• STRs make up 10-15% of the mammalian genome

• STRs are also called “microsatellites”

• STRs are “junk DNA”

Regions of Chromosome Analyzed for DNA

Fingerprinting Often Contain STRs

ACTACTPerson 1 ACTACT

100 ACTrepeats

EcoRI EcoRI

Person 2 ACTACTACTACT

400 ACTrepeats

EcoRI EcoRI

EcoRI fragment from Person 2 is 900bp longer than in Person 1

DNA SynthesisDNA Synthesis

Separatestrands

DNA Replication is Semi-Conservative

AGTCAG

TCAGTC

------

5

53

3

TCAGTC

AGTCAG

5

53

3

AGTCAG

AGTC

------

------

AGTC

TCAGTC

5

53

3

5

5

3

3

AGTCAG

TCAGTC

------

------

AGTCAG

TCAGTC

5

53

3

5

53

3

Add correct bases

Building a Strand of DNA

• DNA polymerase – enzyme that synthesizes DNA

• DNA polymerase can only add nucleotides to the 3´ end of a strand

• DNA polymerase cannot build a new strand without a primer

DNA Polymerase Needs a Primer

3´ 5´ss DNA

+Nucleotides (dNTPs)

+DNA polymerase

=No DNA synthesis

No DNA synthesis

DNA Polymerase Needs a Primer

3´ 5´ss DNA

+Nucleotides (dNTPs)

+DNA polymerase

= DNA synthesis

5´ 3´primer

DNA Polymerase Needs a Primer

3´ 5´ss DNA

+Nucleotides (dNTPs)

+DNA polymerase

= DNA synthesis

5´primer

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

Collect Tissue Sample

How to do DNA FingerprintingThe Big Picture

>1000 cells

RFLP / Southern blot PCR AnalysisPCR Analysis

>20 cells

PCR

Purpose – Quickly make many copies of a region of a DNA molecule

Method – Multiple rounds of DNA replication

Components in PCR reaction – Target DNA, nucleotides, DNA polymerase, and primers

Temperature cycling – DNA replication controlled by temperature…

Temperature Cycling in PCR

Temperature cycling – PCR process uses a machine (thermocycler) in which PCR reaction goes through ~30 cycles of three different temperature changes:

~95ºC – Melting temperature

50-65ºC – Annealing temperature

72ºC – Extension temperature

Polymerase chain reaction (PCR) analysis1). primers are designed to flank the region to be amplified in target DNA2). primers are annealed to denatured DNA3). DNA is synthesized using Taq polymerase (from Thermus aquaticus)4). primers are annealed again and the process is repeated through

20-30 cycles, geometrically amplifying the target sequence5). DNA is analyzed by gel electrophoresis

1).

2).

3).

4).

DNA Technology CSI style

DNA FingerprintingDNA Fingerprinting

Determination of an individual’s unique collection of DNA restriction fragments

Collect Tissue Sample

How to do DNA FingerprintingThe Big Picture

>1000 cells

RFLP / Southern blot PCR AnalysisRFLP / Southern blot

>20 cells

PCR

Purpose – Quickly make many copies of a region of a DNA molecule

Method – Multiple rounds of DNA replication

RFLP AnalysisRFLP Analysis

RFLP – RFLP – RRestriction estriction FFragment ragment LLength ength PPolymorphismolymorphism

-Cut DNA fragments into different sizes w/ -Cut DNA fragments into different sizes w/ restriction enzymerestriction enzyme

-look for matches-look for matches

DNA specimen is extracted from blood or other DNA specimen is extracted from blood or other tissuetissue

Restriction enzymes cut up DNARestriction enzymes cut up DNA DNA fragments are placed in wells made on gelDNA fragments are placed in wells made on gel Electric current is run through gelElectric current is run through gel Fragments separate by sizeFragments separate by size Single chains of separated DNA fragments are Single chains of separated DNA fragments are

blotted onto filter paperblotted onto filter paper Probes bind to complementary DNA fragments Probes bind to complementary DNA fragments

in the samplein the sample Visible bands form on exposed photographic filmVisible bands form on exposed photographic film Film is developed to reveal a DNA fingerprintFilm is developed to reveal a DNA fingerprint

Fingerprint AnalysisFingerprint Analysis

Let’s make some fingerprints!!!Let’s make some fingerprints!!!

Fingerprint TerminologyFingerprint Terminology ridge ending - a ridge that ends abruptly; ridge ending - a ridge that ends abruptly; bifurcation - a single ridge that divides into two ridges; bifurcation - a single ridge that divides into two ridges; lake or enclosure - a single ridge that bifurcates and lake or enclosure - a single ridge that bifurcates and

reunites shortly afterwards to continue as a single ridge; reunites shortly afterwards to continue as a single ridge; short ridge, island or independent ridge - a ridge that short ridge, island or independent ridge - a ridge that

commences, travels a short distance and then ends; commences, travels a short distance and then ends; dot - an independent ridge with approximately equal dot - an independent ridge with approximately equal

length and widthlength and width spur - a bifurcation with a short ridge branching off a spur - a bifurcation with a short ridge branching off a

longer ridge longer ridge crossover or bridge - a short ridge that runs between two crossover or bridge - a short ridge that runs between two

parallel ridges. parallel ridges.

Draw and label Draw and label these three on these three on

your thumbprintyour thumbprint

ridge ending - a ridge that ends abruptly; ridge ending - a ridge that ends abruptly; bifurcation - a single ridge that divides into bifurcation - a single ridge that divides into

two ridges; two ridges; lake or enclosure - a single ridge that lake or enclosure - a single ridge that

bifurcates and reunites shortly afterwards bifurcates and reunites shortly afterwards to continue as a single ridge; to continue as a single ridge;